Exploiting O-GlcNAc dyshomeostasis to screen O-GlcNAc transferase intellectual disability variants

Abstract

O-GlcNAcylation is an essential protein modification catalyzed by O-GlcNAc transferase (OGT). Missense variants in OGT are linked to a novel intellectual disability syndrome known as OGT congenital disorder of glycosylation (OGT-CDG). The mechanisms by which OGT missense variants lead to this heterogeneous syndrome are not understood, and no unified method exists for dissecting pathogenic from non-pathogenic variants. Here, we develop a double-fluorescence strategy in mouse embryonic stem cells to measure disruption of O-GlcNAc homeostasis by quantifying the effects of variants on endogenous OGT expression. OGT-CDG variants generally elicited a lower feedback response than wild-type and Genome Aggregation Database (gnomAD) OGT variants. This approach was then used to dissect new putative OGT-CDG variants from pathogenic background variants in other disease-associated genes. Our work enables the prediction of pathogenicity for rapidly emerging de novo OGT-CDG variants and points to reduced disruption of O-GlcNAc homeostasis as a common mechanism underpinning OGT-CDG.

Keywords: O-GlcNAc; OGT; OGT-CDG; neurodevelopment.

Graphical abstract

Figure 1

Fusion of sfGFP to endogenous OGT does not disrupt O-GlcNAc homeostasis (A) Schematic of the CRISPR knockin strategy used to fuse sfGFP to the C terminus of endogenous Ogt in mESCs. (B) Immunoblot of proteins extracted from CRISPR-engineered OGT-sfGFP and untreated wild-type mESCs, using antibodies against OGT and GFP (see also Figure S1). (C) Left: OGT and O-GlcNAc (RL2) levels in OGT-sfGFP mESCs compared to untreated wild-type mESCs, with PGK1 as a loading control. Right: O-GlcNAc and OGT levels normalized to PGK1 (n = 3 independent experiments). Error bars represent the standard error of the mean (SEM). p values (unpaired t test): OGT = 0.10, O-GlcNAc = 0.17.

Figure 2

OGT-sfGFP levels/fluorescence respond to the pharmacological disruption of O-GlcNAc homeostasis (A) Immunoblot showing OGT and O-GlcNAc (RL2) levels in OGT-sfGFP mESCs after 24-h treatment with 10 μM OSMI-4b or TMG, alongside a 0.1% DMSO vehicle control and an untreated control. PGK1 served as a loading control. Statistical analysis was performed on the right using urdinary one-way ANOVA (n = 3 independent replicates), with error bars representing mean ± SEM. (B) Density plots of gated live singlet OGT-sfGFP mESCs after 10 μM OSMI-4b or TMG treatments (see gating strategy in the no-transfection section of Figure S2). OGT-sfGFP fluorescence is displayed on the x axis, and propidium iodide (PI) fluorescence on the y axis. (C) Overlay of sfGFP histograms showing gated live singlet OGT-sfGFP mESCs after 10 μM OSMI-4b or TMG treatments, with OGT-sfGFP fluorescence on the x axis and cell count on the y axis. (D) Median sfGFP fluorescence values from the sfGFP histogram (Figure 2C) were extracted for each sample and subjected to statistical analysis via ordinary one-way ANOVA (n = 3 independent replicates), with error bars indicating mean ± SEM. (E) Images of OGT-sfGFP mESCs after 10 μM OSMI-4b or TMG treatments, captured in both bright-field (BF) and sfGFP fluorescence channels using an ImageStream flow cytometer. Statistical significance for all experiments is denoted as follows: ^∗ for adjusted p < 0.05, ^∗∗ for p < 0.01, ^∗∗∗∗ for p < 0.0001, and ns for p > 0.05.

Figure 3

Endogenous OGT-sfGFP is a readout for the activity of exogenous OGT variants (A) Illustration of plasmids used in the study: a wild-type OGT plasmid without fluorescent labeling (OGT-WT), a mock control plasmid with a FLAG tag linked to mTagBFP2 via the P2A linker, and mTagBFP2-OGT plasmids, where OGT or its variants are linked to mTagBFP2 via the P2A linker. (B) Density plots of gated live singlet OGT-sfGFP mESCs after transfection with the unlabeled OGT-WT plasmid, fluorescently labeled mTagBFP2-OGT-K842M or mTagBFP2-OGT-WT plasmids, and the mock control. The x axis shows OGT-sfGFP fluorescence intensity, and the y axis displays mTagBFP2 fluorescence. (C) Selection of transfected mTagBFP2⁺ cells. A universal gate was applied across all samples to select OGT-sfGFP cells expressing mTagBFP2 (see plasmid-transfection section in Figure S2), with untransfected cells used as a control to define the gate. (D) Overlay of sfGFP histograms for gated mTagBFP2⁺ cells. Clear separations in sfGFP fluorescence were observed between cells transfected with mTagBFP2-OGT-WT, mTagBFP2-OGT-K842M, and the mock control plasmids. (E) Statistical analysis of OGT-sfGFP fluorescence in gated mTagBFP2⁺ mESCs following overexpression of mTagBFP2-OGT-WT, mTagBFP2-OGT-K842M, and the mock control. Each data point represents the median sfGFP fluorescence for the corresponding mTagBFP2⁺ cells. Ordinary one-way ANOVA was performed (n = 3 independent replicates); ^∗∗∗∗ denotes an adjusted p value <0.0001. Error bars represent mean ± SEM.

Figure 4

Changes in OGT-sfGFP fluorescence predict OGT-CDG variant pathogenicity (A) Schematic of all OGT variants used in this study. Variants in blue represent the eight most frequent OGT variants from the gnomAD database (gnomAD variants; Table S2). Variants in pink are previously reported pathogenic OGT-CDG variants, while those in orange are three newly identified ID-associated OGT variants reported in this study. All variants are tagged with mTagBFP2 via the P2A linker for transfection. (B–D) Statistical analysis of OGT-sfGFP fluorescence in transfected mTagBFP2⁺ cells. Each OGT variant was transfected into OGT-sfGFP mESCs at least six times across different passages and days. Each data point represents the median OGT-sfGFP fluorescence from an independent transfection sample, normalized to the median fluorescence of the K842M mutant transfected sample. Analysis of gnomAD variants is shown in (B), previously reported pathogenic OGT-CDG variants in (C), and the three newly identified potential OGT-CDG variants in (D). Error bars represent mean ± SEM (n ≥ 6). Ordinary one-way ANOVA was performed for each graph, with symbols indicating significance: ^∗∗ for adjusted p < 0.01, ^∗∗∗∗ for p < 0.0001, and ns for p > 0.05. A panel of representative density plots for mTagBFP2⁺ cell selection across all transfected variants is presented in Figure S4.

Figure 5

O-GlcNAc feedback regulation involves the transcriptional coordination of OGT and OGA (A) Representative density plots for sorting mTagBFP2⁺ cells following transfection with a subset of plasmids, including the mock control, wild-type OGT, the inactive K842M OGT mutant, the gnomAD I279V variant, and the pathogenic N648Y OGT-CDG variant. The sorted mTagBFP2⁺ cells were then used for immunoblotting (B and C) and RT-qPCR (D) analysis. (B) Representative western blots from lysates of sorted mTagBFP2⁺ cells, blotted for mTagBFP2, GFP, OGT, O-GlcNAc (RL2), and OGA, with PGK1 as a loading control. (C and D) Statistical analysis of western blot (C) and RT-qPCR (D) results. Ordinary one-way ANOVA was performed for all experiments (n = 3 independent replicates). Error bars represent mean ± SEM, with significance indicated as follows: ^∗ for adjusted p < 0.05, ^∗∗ for p < 0.01, ^∗∗∗ for p < 0.001, ^∗∗∗∗ for p < 0.0001, and ns or no mark for non-significant results (adjusted p > 0.05).